SeqShop: Calling Your Own Genome, December 2014

Login instructions for seqshop-server

Login to the seqshop-server Linux Machine

This section will appear redundantly in each session. If you are already logged in or know how to log in to the server, please skip this section

1. Login to the windows machine

• The username/password for the Windows machine should be written on the right-hand monitor

Start xming so you can open external windows on our Linux machine

• Start->Enter "Xming" in the search and select "Xming" from the program list
• Nothing will happen, but Xming was started.
View Screenshot

Open putty

• Start->Enter "putty" in the search and select "PuTTY" from the program list
View Screenshot

Configure PuTTY in the PuTTY Configuration window

• Host Name: seqshop-server.sph.umich.edu
View Screenshot



• Setup to allow you to open external windows:
• In the left pannel: Connection->SSH->X11
• Add a check mark in the box next to Enable X11 forwarding
View Screenshot



• Click Open
• If it prompts about a key, click OK

Enter your provided username & password as provided

You should now be logged into a terminal on the seqshop-server and be able to access the test files.

• If you need another terminal, repeat from step 3.

Login to the seqshop Machine

So you can each run multiple jobs at once, we will have you run on 4 different machines within our seqshop setup.

• You can only access these machines after logging onto seqshop-server

3 users logon to:

ssh -X seqshop1

3 users logon to:

ssh -X seqshop2

2 users logon to:

ssh -X seqshop3

2 users logon to:

ssh -X seqshop4

Setup

The snpcall pipeline will run overnight, but you'll want to log out.

How do I leave something running on the server even if I log out?

One solution is screen!

How do I use screen?

Before running your command, you need to start screen:

screen

As it says, press Space or Return.

• It should now look basically the same as your normal command line.

Scrolling problems when using screen?

If you want to scroll and screen doesn't scroll like you normally would?

• Type Ctrl-a Esc and you should be able to scroll up with your mouse wheel
  • Or at least that is what I do from my Linux machine - (sorry I'm typing this up/testing these commands from Linux and not windows, so can't test it out)

Set these values. Also, be sure to specify your sample name instead of SampleXX

export SAMPLE=SampleXX
source /net/seqshop-server/home/mktrost/seqshop/setupSS.txt

See the settings you just used:

cat /net/seqshop-server/home/mktrost/seqshop/setupSS.txt

Shows you:

export GC=/net/seqshop-server/home/mktrost/seqshop/gotcloud

export OUT=~/$SAMPLE/output

List of BAMs

The list of BAMs has already been created (just 1 BAM, your sample).

• But it is simply SAMPLE\tBAM_name, so easy to figure out

cat $SAMPLE/output/bam.list

SampleXX SampleXX/output/bams/SampleXX.recal.bam

• Relative path, so assumes running from your home directory (I prefer absolute paths, but for simplicity of the workshop, we just use relative path).

Configuring SnpCall

cat $SAMPLE/gotcloud.conf

You will see something like this:

# Cluster Settings
BATCH_TYPE = 
BATCH_OPTS = 

OUT_DIR = Sample13/output

# Align Settings
MAP_TYPE = BWA_MEM
BWA_THREADS = -t 24
FASTQ_LIST = fastq.list

# SNP Call Settings
UNIT_CHUNK = 20000000      # Chunk size of SNP calling : 20Mb
VCF_EXTRACT = /net/seqshop-server/home/mktrost/seqshop/singleSample/snpOnly.vcf.gz
MODEL_GLFSINGLE = TRUE
MODEL_SKIP_DISCOVER = FALSE
MODEL_AF_PRIOR = TRUE

EXT_DIR = /net/seqshop-server/home/mktrost/seqshop/singleSample/ext
EXT = $(EXT_DIR)/ALL.chrCHR.phase3.combined.sites.unfiltered.vcf.gz $(EXT_DIR)/chrCHR.filtered.sites.vcf.gz

Running SnpCall

${GC}/gotcloud snpcall --conf $SAMPLE/gotcloud.conf --numjobs 6

• Only need the configuration & number of threads, rest is specified within the configuration.

Run 4 jobs on our mini-cluster

$GC/gotcloud snpcall --conf ~/personal/gotcloud.2x.conf --numjobs 4 --batchtype mosix --batchopts "-j10,11,12,13"

• --batchtype says to use mosix (our cluster system)
• --batchopts tells mosix the options to run with
  • for mosix, -j10,11,12,13 says to run on nodes 10, 11, 12, & 13 - the names of the 4 nodes on our mini-cluster

Exome

To speed things up, I extracted only exome regions from 100 1000g low coverage BAMs.

Let's create a new bam info file with your BAM combined with those BAMs.

cp $ALIGN_OUT/bam.index $EXOME_OUT/bam.exome.index

Now add the exome BAMs to your new bam list:

cat $IN/exome/bam.exome.index >> $EXOME_OUT/bam.exome.index

Verify you have 101 lines in your list:

wc -l $EXOME_OUT/bam.exome.index

We are going to run on the cluster, so edit the first line of $EXOME_OUT/bam.exome.index to give the cluster path to your info file.

nedit $EXOME_OUT/bam.exome.index

Replace the /home on the first line with /net/seqshop-server

Locate your gotcloud.2x.conf (probably at: ~/personal/gotcloud.2x.conf) and open it in your favorite editor:

nedit  ~/personal/gotcloud.2x.conf

Replace all occurrances of

/home with /net/seqshop-server

This is so you can run on the mini-cluster we have and can run more jobs at once

Update OUT_DIR & BAM_INDEX to:

OUT_DIR = $(IN_DIR)/output.exome
BAM_INDEX = $(OUT_DIR)/bam.exome.index

Update your gotcloud configuration file to indicate exomes:

# Specify the path to the regions we want to call
UNIFORM_TARGET_BED = $(REF_DIR)/20130108.exome.targets.nochr.bed

# We do not want any off target bases
OFFSET_OFF_TARGET = 0

WRITE_TARGET_LOCI = TRUE
TARGET_DIR = target

Remove CHRS = 20

Since it would take a while to run all 101 samples, I already ran the first step for the 100 1000G samples. We will "trick" GotCloud into thinking you already ran them by copying them into your output directory.

cp -r $IN/exome/glfs $EXOME_OUT/.

Run 4 jobs on our mini-cluster

$GC/gotcloud snpcall --conf ~/personal/gotcloud.2x.conf --numjobs 4 --batchtype mosix --batchopts "-j10,11,12,13"

• --batchtype says to use mosix (our cluster system)
• --batchopts tells mosix the options to run with
  • for mosix, -j10,11,12,13 says to run on nodes 10, 11, 12, & 13 - the names of the 4 nodes on our mini-cluster

Log Out

Want to log out and leave your job running?

In the screen window, type:

Ctrl-a d

(Hold down Ctrl and type 'a', let go of both and type 'd')

• This will "detach" from your screen session while your alignment continues to run.

If you have not detached from screen:

Ctrl-a d

exit PuTTY

Logging Back in to Check Jobs

How do you log back into screen tomorrow?

screen -r

This will resume an already running screen.